The present invention relates generally to fluorogenic compounds, including novel fluorogenic compounds suitable for use in biological assays, and to methods of using the flourogenic compounds in biological assays.
Fluorogenic and chromogenic enzyme substrates find broad utility in biological detection assays. Many of these substrates are formed by covalently linking a fluorescent or chromophoric dye to a biological molecule that is specific to an enzyme being investigated. Subsequent cleavage of the covalent linkage by the enzyme releases the dye, allowing the fluorescent or calorimetric properties of the dye to be detected visually or measured spectrophotometrically.
The challenge in using this method is finding a fluorophore or chromophore that can satisfy a wide range of conditions for the biological assay of interest. For instance, the fluorescence or the color of the cleaved product should preferably vary from the uncleaved substrate, the background fluorescence and color of the biological sample should not interfere with the detection of the cleaved product, the substrate should be stable to heat and light under the conditions required for the assay, and the substrate should not interfere with the biological activity of the enzyme.
Commonly used substrates include fluorogenic synthetic enzyme substrates derived from coumarin derivatives 4-methylumbelliferone (4-MU) or 7-amino-4-methylcoumarin (7-AMC). MU derivatives have the following structure: 
The popularity of these substrates can be ascribed to availability of a wide range of enzyme cleavable R groups. Typical R groups frequently used are esters, monosaccharides, disaccharides, and phosphates. In addition, the fluorogenic synthetic enzyme substrates are popular because of their noncarcinogenicity, ease of visual detection of the products of enzyme activity with UV light sources, autoclave stability, and availability of suitable fluorometers for measurement of fluorescence in both tube and multiwell panels. Enzyme cleavage releases 4-MU, giving rise to fluorescence associated with the 4-MU anion (excitation wavelength of 365 nm, emission wavelength 440 nm). Aryl peptides of 7-AMC are also frequently used as fluorogenic enzyme substrates. The released 7-AMC shows a blue fluorescence (excitation wavelength 370 nm, emission wavelength 440 nm).
Release of 4-MU and 7-AMC can be detected visually as a blue fluorescence when irradiated with a long wavelength UV lamp (for example, xcex=360xc2x120 nm). However, at these excitation and emission wavelengths, it is quite common for the biological sample to emit a significant background fluorescence of its own. In other cases, materials employed in making the plate, tube or polymeric support of an assay format can emit a significant background fluorescence. In these cases, low amounts of enzyme activity cannot be detected above background. This common problem adversely affects the sensitivity and speed of many enzyme-linked biological assays.
In contrast, at longer excitation and longer emission wavelengths, background fluorescence drops off dramatically. Therefore, it would be advantageous to develop a class of fluorogenic enzyme substrates that absorb and emit at longer wavelengths (i.e., are red-shifted) than those of the 4-MU- and 7-AMC-based derivatives, without losing the desirable properties of these dyes.
Alternative detection methods do exist. For example, esters, monosaccharides, disaccharides, and phosphates of o-nitrophenol (ONP) or p-nitrophenol (PNP) are frequently used as colorimetric enzyme substrates. Release of nitrophenol gives rise to an increase in absorbence at 405 nm and appearance of a yellow color. The absorbence of these products can be detected using, e.g., a GaN LED source. However, these products are not fluorescent, therefore the sensitivities are far less than 4-MU and 7-AMC derivatives. Aryl peptide derivatives of p-nitroanaline (p-NA) give similar appearance of yellow color. In this case, a substantial increase in sensitivity can be achieved by reacting the p-NA with a diazo dye, yielding blue to dark purple colors. Esters of indoxyl or 5-bromo-4-chloro-3-indolyl give rise to enzyme induced release of indoxyl, to form a blue color, but still do not result in sufficient fluorescence output that allows for more sensitive measurements.
Esters, monosaccharides, disaccharides, and phosphates of fluorescein (FL) and rhodamine (RH) dyes have been developed as fluorogenic enzyme substrates. Release of FL gives rise to an increase in fluorescence associated with the FL anion (excitation wavelength 490 nm, emission wavelength 514 nm). Release of RH gives rise to an increase in fluorescence associated with the RH anion (excitation wavelength 499 nm, emission wavelength 521 nm). The FL and RH anion fluorescences can easily be detected using bulky gas lasers as excitation sources, but there is currently no commercially available solid state light source for these materials. In addition, these classes of enzyme substrates are not typically autoclave stable and therefore are not appropriate for many applications. Finally, the Stokes shift (the difference between the absorbence and emission wavelength) for these materials is far less than that for the 4-MU and 7-AMC materials, requiring the use of more specialized optics to separate the emission signal from the excitation signal.
One particularly relevant application for enzyme substrate indicators is in the detection and differentiation of bacteria. Growing microcolonies will often secrete extracellular enzymes that can convert upwards of a million fluorescent indicator molecules per enzyme molecule. Because the fluorescence detection method is extremely sensitive, this provides a method to amplify the signal from a growing microcolony so that it can be detected in a shorter period of time. For example, a growing microcolony might be detected in 4 to 6 hours using fluorogenic enzyme substrates, whereas the microcolony may be detected in 24 to 48 hours using conventional chromogenic enzyme substrates. This would offer great benefit in the food processing industry, as contaminants could be discovered in eight hours or less.
An example where such methods would be useful is detection of E. coli or coliform. E. coli is an important indicator of fecal contamination in environmental and food samples, while coliform count is an important indicator of bacteriological contamination. In the quality control of water and food, it is highly important to examine both coliforms and E. coli. Testing procedures for coliforms commonly employ a 4-MU derivative specific for detecting xcex2-D-galactosidase (xcex2-Gal) activity. This substrate is 4-methylumbelliferyl-xcex2-D-galactoside (MUGal), which is hydrolyzed by xcex2-Gal, liberating blue fluorescent 4-MU. Testing procedures for E. coli commonly employ a 4-MU derivative specific for detecting xcex2-D-glucuronidase (xcex2-Gud) activity. This substrate is 4-methylumbelliferyl-xcex2-D-glucuronide (MUGud), which is hydrolyzed by xcex2-Gud, again liberating 4-MU. For selective detection of E. coli in primary isolation media, it is common to perform an aerobic incubation in a selective growth medium that inhibits growth of gram-positive strains. In this way, xcex2-Gud activities from strains other than E. coli are suppressed. Additionally, incubation at 44xc2x0 C. and detection of gas formation help in exclusive detection of E. coli. 
Several MUGud and MUGal testing procedures have been employed for identifying and enumerating total coliforms and E. coli. These include most probable number, membrane filtration, presence absence test, agar plate, microtitration plate, paper strip, and related techniques. Because of the thermal stability of these dyes to autoclave, they can be incorporated directly into the growth media before autoclave sterilization. This is an important advantage for commercial test kits, which are sterilized and packaged in the factory.
In a related application, it has been desirable to use the autoclave stability of 4-MU derivatives to develop a biological indicator for monitoring the effectiveness of a sterilization procedure. In this case, a product sold under the trade name Attest(trademark) Rapid Readout Biological Indicator (3M, St. Paul, Minn.) employs Bacillus subtilis or Bacillus stearothermophilus spores as the active biological agent. These are very resilient spores that are difficult to kill unless the sterilization conditions are rigorous. One way to test for them is to prepare a device containing Bacillus stearothermophilus spores in one compartment, and 4 -methylumbelliferyl-xcex1-D-glucoside (xcex1-MUGlc) in a second compartment. The device is exposed to autoclave conditions, after which the spores and the enzyme substrate indicator are mixed. If the spores are killed, no 4-MU fluorescence is detected. If the spores are not killed, 4-MU fluorescence is observed to increase. Correspondingly, Bacillus subtilus activity is detected in a similar format using 4-methylumbelliferyl-xcex2-D-glucoside (xcex2-MUGlc) as the enzyme substrate indicator.
Another important application area involves the use of fluorogenic enzyme substrates in selective binding assays for clinical diagnostic and high throughput screening applications. In this format, a target biomolecule is detected using a primary or secondary detection reagent (e.g. an antibody or a DNA probe) that has been conjugated to an enzyme. The primary reagent binds selectively to the target. In certain cases, a secondary reagent is added that binds selectively to the primary reagent. After removing unbound reagents, the signal associated with the conjugated enzyme is developed by adding a suitable fluorogenic or chromogenic enzyme substrate. Again, the enzyme acts as an amplifier by cleaving one million or more dye molecules per enzyme molecule.
ELISA assays (Enzyme-Linked Immuno-Sorbant Assays) are one of the most common immunoassay formats used in clinical and research applications. In this case, antibodies bound to a solid support are used to selectively capture the biological target. Next, an enzyme conjugated antibody reporter probe is introduced that binds to the captured targets, forming a sandwich. After rinsing off unbound reporter, fluorescent indicator is introduced to develop the signal. A related assay for nucleic acid detection employs oligonucleotide capture probes bound to a solid support, and enzyme conjugated oligonucleotide reporter probes added to form the sandwich. In addition, enzyme-conjugated detection reagents can also be used to localize a signal in cells or tissue.
Homogeneous immunoassay techniques are generally more rapid and convenient than their heterogeneous counterparts. U.S. Pat. No. 4,259,233 teaches the use of xcex2-galactosyl-umbelliferone-labeled protein and polypeptide conjugates in immunoassays. In these assay formats it is desirable to conjugate a fluorogenic enzyme substrate to a macromolecular substrate identical to the biological target molecule under assay. In this case, the sample target and conjugated target (having the fluorogenic enzyme substrate) compete for binding to a fixed pool of antibodies. Once the antibodies bind to the conjugated target, they inhibit access of added xcex2-Gal enzyme, and the fluorogenic enzyme target is protected from cleavage. As the amount of sample target increases, the number of antibodies available to protect the conjugate target decreases, and the fluorescent signal from enzymatically cleaved conjugate increases.
Enzyme fragment recombination offers an alternative approach to homogenous assays. Genetically engineered fragments of xcex2-galactosidase enzyme derived from E. coli are known to recombine in vitro to form active enzyme. This reaction can be used as a homogeneous signaling system for high-throughput screening. In this type of assay, a biological ligand such as a drug is conjugated to one of the enzyme fragments. The ligand alone does not adversely affect enzyme fragment recombination. However, if an antibody, receptor or other large biomolecule is added that specifically binds to the ligand, enzyme fragment recombination is sterically impeded and enzyme activity is lost. Receptor binding efficiency to the ligand is determined from the kinetics of enzymatic cleavage of added fluorogenic enzyme substrate. Again, enzymatic amplification is achieved by measuring the signal produced by the action of enzyme on fluorogenic compounds such as MUGal.
Thus, there is a need to develop new enzyme-linked assay formats that employ very small sample sizes, such as assays performed in microwell arrays, on microfluidic chips, and on the surface of micron scale bead carriers. There is also a need to develop rapid, compact, high-sensitivity readers that can detect and quantify the enzyme-linked fluorescent response. It would be highly desirable to interrogate the fluorescent response in these formats using inexpensive and compact solid state light sources, instead of bulky lamp sources. However, there is currently no solid state light source that can excite the 4-MU and 7-AMC products. It would be advantageous to develop a class of fluorogenic enzyme substrates that are spectrally compatible with a solid state light emitting diode or laser diode source. Also, in some of these applications, a need remains for improved fluorogenic enzyme substrates that are red-shifted, LED or laser diode compatible, and autoclave stable.
The present invention is directed to coumarins bearing, at the 7-position, an enzymatically-cleavable group and, at the 3-position, a 5-membered heterocyclic ring, wherein the coumarins are useful as fluorogenic enzyme substrates (FES) conveying improved properties for rapid microbial detection applications. Specifically, the coumarin-based FES so modified are substantially red-shifted in absorbence with respect to traditional 4-MU derivatives, making them compatible with the use of GaN LED and laser diode excitation sources having xcexemission in the range of 390 to 540 nm. Furthermore, these molecules are typically autoclave stable, making them compatible with a variety of biological indicator assay formats for sterilization monitoring, sterilization processes applied in the manufacture of biological assay kits and materials, and laboratory protocols for sterilization of growth media and other materials used in biological research. In addition, in certain implementations, the pKa of the enzymatically cleaved products can be modulated in the range of 6.1 to 7.6 for a wide range of biological assays which perform optimally at or near physiological pH. The modulation is performed based upon the choice of the five membered heterocyclic substituents.
Accordingly, the present invention includes a fluorogenic compound of Formula (I), provided below: 
wherein Q is selected from the group consisting of a glycone, a glycosyl phosphate, an ester, and a peptide; each R2 independently is a sterically non-interfering group; R3 is an electron withdrawing or non-electron withdrawing group; Z is O or NR5, wherein R5 is hydrogen or a hydrocarbyl-containing group; Y and Y1 independently are O, S, NHx, or CHy where x is 0 or 1 and y is 1 or 2, with the proviso that at least one of Y and Y1 is O, S, or NHx; and each R4 independently is selected from the group consisting of hydrogen, halogen, a C1-C20 alkyl, a C1-C20 alkoxy, a C3-C18 cycloalkyl, a C6-C18 aryl, a C6-C18 aryloxy, a C6-C18 hydroxyaryl, a C6-C18 arylcarboxy, a C6-C18 carboxyaryl, a C2-C18 alkenyl, a C1-C20 hydrocarbylamino, a C6 -C18 arylamino, a C6-C18 aminoaryl, a C2-C20 di(hydrocarbyl)amino, a heterocyclic group having at least three ring atoms, carboxyl, carboxamide, ester, keto-alkyl ester, sulfonic acid, amino, a pendant biological ligand such as a protein or polypeptide of molecular weight between 130 and 10,000,000, and, more specifically, an immunoglobulin, and a group of the formula (CH2A)aE in which A is O, NH, or a single bond, E is a functional group that includes active hydrogen, and a is a whole number from 1 to 100, preferably from 1 to 25, and more preferably 1 to 10; or both R4 groups together with the carbon atoms to which they are attached form a 5- or 6-membered aromatic ring which optionally can have one or more R4 groups attached; or a salt thereof. Where this molecule is to be covalently attached to a support or a biological ligand, at least one of R2, R3, and R4 typically includes a group having an active hydrogen.
In one embodiment, the present invention teaches a class of coumarin based fluorogenic enzyme substrates (FES). When the enzymatically hydrolyzable group on the fluorogenic substrate is selectively hydrolyzed by an enzyme, the properties of the fluorophor change in such a way that the enzyme activity can be detected visually through a color change or through a change in fluorescence. Further, the enzyme activity can be quantified for a particular sample by fluorometric or colorimetric analysis.
In a preferred embodiment, substituent groups and their positions on the coumarin ring have been chosen so as to ensure that the excitation (i.e. absorption) maximum of the enzymatically cleaved product is centered at a wavelength greater than 380 nm. This allows the ionophore of the present invention to be used with certain solid state light sources such as, for example, blue LEDs and lasers. Substituent groups and their positions are also preferably chosen to keep the pKa of the cleaved product below pH 7.6 so that the deprotonated fluorogenic cleavage product is present in detectable amounts when using the fluorogenic substrates in biological assays. Finally, substituent groups and their positions are preferably chosen to provide the option for covalent attachment to polymeric or ceramic supports, oligomers, biopolymers, biological ligands such as proteins and polypeptides, and the like. Preferably, the material to which the indicator is attached is chosen to support uniform and reproducible enzymatic response.
Another aspect of the present invention is found in a composite structure having a polymeric support and the fluorogenic compound of formula (I) bonded to the support through at least one of R2, R3, and R4 by means of either a bond or a linking group, the linking group having functionalities at both ends, wherein the functionality at one end of the linking group is complementary to the functionality of R2, R3 or R4 and the functionality at the other end is complementary to a functional group on the support.
The polymeric support can be in the form of a sensor disk, a polymeric fiber or microwell, or a component on the surface of a carrier bead suitable for interrogation by, for example, flow cytometry or microscopy. Suitable coupling agents for covalent attachment are described in U.S. Pat No. 5,053,520, which is herein incorporated in its entirety by reference. Homobifunctional and/or heterobifunctional coupling agents are described in PCT Pat. App. Nos. WO 96/07268 and WO 96/10747, also incorporated herein by reference in its entirety.
Still another aspect of the present invention is found in a fluorogenic macromolecular conjugate having a molecular ligand or macromolecular ligand and the fluorogenic compound of Formula (I) covalently linked through at least one R2, R3, and R4 by means of one of a bond and a linking group, the linking group having functionalities at both ends, wherein the functionality at one end of the linking group is complementary to the functionality of R2, R3, or R4, and the functionality at the other end is complementary to a functional group on the ligand.
In a further aspect, the present invention provides a sensing system that comprises a sensing element and an excitation assembly, wherein the sensing element comprises at least one of the fluorogenic enzyme substrates and a fluid handling architecture structured and adapted to support mixing or otherwise intimate interaction of one or more enzyme-containing samples with at least one of the fluorogenic enzyme substrates to enable enzymatic reaction to form a cleaved fluorescent product which, when exposed to light of a wavelength range centered around xcex1, is capable of emitting light of a wavelength range centered around xcex2, wherein xcex2 is at least 10 nm greater than xcex1, xcex1 is at least about 380 nm, more preferably at least about 425 nm, and most preferably at least about 450 nm. In one preferred embodiment, the excitation assembly comprises solid state GaN, InGaN, ZnSe, or SiC LED or laser diodes which excite the sensing element(s) at or near wavelength xcex1 for a time sufficient for the cleaved product to emit visible light of wavelength xcex2.
In specific implementations, a detection assembly is positioned and adapted to detect the intensity or location of emitted signal(s) centered at xcex2 from the sensing element. The output from the detection assembly is typically converted to a digital signal by an analog to digital (A/D) converter and transmitted to the processor assembly. A processor assembly is positioned and adapted to process and analyze the emitted signal(s) in determining the concentration, location, or enumeration of biomolecules, bio-macromolecules, or microorganisms. This processor assembly may be part of a stand-alone unit or may be part of a central computer or local area network. Optionally, the processor assembly may contain a relational data base which correlates the processed data for each sensing element with corresponding identifiers for samples or articles, e.g., a food sample, a drug sample, a clinical sample, a sterilized article, etc.
In one embodiment, the excitation and detection assemblies are integrated using bifurcated fiber-optics that direct light of wavelength xcex1 from an LED to one or more sensing elements or simultaneously to sensing elements and a reference channel. Return fibers direct emitted signals of wavelength xcex2 from the sensing elements and reference channels to optical detectors for analysis by the processor.
In a second embodiment, the excitation and detection assemblies are integrated using a beam splitter assembly and focusing optical lenses that direct light of wavelength xcex1 from an LED to a single location on a sensing element and direct emitted light of wavelength xcex2 from the sensing element to an optical detector for analysis by the processor. In this embodiment, the measurement can be made at one point on the sensing element, as for example in detecting emission from a cuvette, sensor disk, or a channel within a microfluidic chip. Also, the measurement can be made at multiple locations on the sensing element by moving or raster scanning the sensing element including a point light source and focusing optical lenses with respect to one another, as for example in detecting emission from multiple growing microcolonies on a planar growth medium configured to absorb a fluid sample containing viable microorganisms and to support the growth of the viable microorganisms, such as Petrifilm(trademark) (3M, St. Paul, Minn.). In this case, the processor assembly optionally analyzes data from these multiple points on the sensing element to determine the concentration, location, or enumeration of the biomolecules, bio-macromolecules, or microorganisms corresponding to that sample.
In a third embodiment, the excitation and detection assemblies are integrated as a scanner comprising a bank of LED""s or other suitable light sources adjacent to a CCD camera chip. The LED""s direct light of wavelength xcex1 to an area on the sensing element. The CCD camera collects emitted light of wavelength xcex2 and provides a fluorescent image of the defined area to the processor for analysis. In this case, the processor is adapted to interpret the image in determining the concentration, location, enumeration or identity of the biomolecules, bio-macromolecules, or microorganisms corresponding to that sensing element. In some applications, it is desirable to print biological components in a predefined pattern such as a logo, indicia, barcode, or array and to interpret the resulting image after exposure to the fluorogenic enzyme substrate. For example, a microwell array may contain a panel of different drug candidates to be screened by an assay that involves using the fluorogenic enzyme substrate as an indicator. Or, a panel of several biological receptors may be printed on a support, exposed to a biological sample, and developed using a fluorogenic enzyme substrate. The resulting fluorescent image can be analyzed by the processor using image recognition software designed to orient, scale, and enhance the image so that it conforms to a standard form that can be interpreted.
In a further aspect, the present invention provides a method of detecting the presence of enzymatic activity comprising the steps of (a) contacting a fluorogenic enzyme substrate with an enzyme-containing medium and allowing or providing a means for the enzyme to act on the enzyme substrate (e.g., through diffusion) to release some or all of the cleaved fluorescent product, which when exposed to light of a wavelength range centered around xcex1 is capable of emitting light of a wavelength range centered around xcex2, wherein xcex2 is at least 10 nm greater than xcex1, xcex1 is at least about 380 nm, more preferably at least about 425 nm, and most preferably at least about 450 nm; and (b) interrogating the cleaved product with light of a wavelength range centered around xcex1 for a time sufficient for the product to emit visible light of wavelength xcex2 which is collected and detected. By means of suitable analysis algorithms, the amount, location, or rate of change of the emitted light can be correlated with the concentration or enumeration of biomolecules, biomacromolecules, or microorganisms in the enzyme-containing medium. Alternatively, the fluorogenic enzyme substrate can be located throughout the growth medium and the enzyme-containing sample can be contacted therewith. Diffusion of the enzyme into the medium allows the enzyme to act on the enzyme substrate to release some or all of the cleaved fluorescent product, and the light emitted therefrom is subsequently detected and analyzed, as described above.